Isotope exchange at equilibrium indicates a steady state ordered kinetic mechanism for human sulfotransferase.
Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates biosignaling molecular biological activities and detoxifies hydroxyl-containing xenobiotics. The universal sulfuryl group donor for SULTcatalyzed sulfation is adenosine 3'-phosphate 5'-phosphosulfate (PAPS). The reaction products are a sulfated product and adenosine 3',5'-diphosphate (PAP). Although the kinetics has been reported since the 1980s,SULT-catalyzed reaction mechanisms remain unclear. Human SULT1A1 catalyzes the sulfation of xenobiotic phenols and has very broad substrate specificity. It has been recognized as one of the most important phase II drug-metabolizing enzymes. Understanding the kinetic mechanism of this isoform is important in understanding drug metabolism and xenobiotic detoxification. In this report, we investigated the SULT1A1-catalyzed phenol sulfation mechanism. The SULT1A1-catalyzed reaction was brought to equilibrium by varying substrate (1-naphthol) and PAPS initial concentrations. Equilibrium constants were determined. Two isotopic exchanges at equilibrium ([14C]1-naphthol <=>[14C]1-naphthyl sulfate and[35S]PAPS<=>[35S]1-naphthyl sulfate) were conducted. First-order kinetics, observed for all the is otopic exchange reactions studied over the entire time scale that was monitored, indicates that the system was truly at equilibrium prior to addition of an isotopic pulse. Complete suppression of the 35S isotopic exchange rate was observed with an increase in the levels of 1-naphthol and 1-naphthyl sulfate in a constant ratio,while no suppression of the 14C exchange rate was observed with an increase in the levels of PAPS and PAP in a constant ratio. Data are consistent with a steady state ordered kinetic mechanism with PAPS and PAP binding to the free enzyme.
Tyapochkin
Eduard
E
Department of Physiological Sciences, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Cook
Paul F
PF
Chen
Guangping
G
D002250:Carbon Radioisotopes / Q000378:metabolism; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D019800:Phenol / Q000737:chemistry / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry / Q000378:metabolism*; D013431:Sulfates / Q000737:chemistry / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism*
D002250:Carbon Radioisotopes; D013431:Sulfates; D019800:Phenol; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases